A. FIELD OF THE INVENTION
The invention is generally in the field of quantitative assay of gastric cancer in order to provide early diagnosis in differentiating between malignant and benign tumors of the gastric mucosae.
B. DESCRIPTION OF THE PRIOR ART
The antigen and the antibody which form a complex in the diagnosis of gastric cancer is described in my prior U.S. Pat. No. 4,219,539. This antigen, as described in my earlier patent, is reported in Cancer Research, Vol. 33, January 1973 and the antigen reacts immunochemically to give one precipitin line on double diffusion in agar gel. The qualitative test described in the patent is effective with concentrations of tumor globulin from gastric aspirate of the order of 250 micrograms per ml of samples taken from the gastric juice of the human patient at risk satisfies three additional diffusion criteria.
It is known in the prior art to employ separation procedures using chromatography in order to concentrate the protein antigen and thereby obtain greater sensitivity for a quantitative assay using enzyme linked immunosorbent assay which is hereinafter referred to as ELISA.
The U.S. Pat. No. 4,269,765, to Matsuda, describes a method of preparing specific immune serum from the fluids of cancer patients useful in diagnosis of cancer in which peritoneal fluid from stomach cancer patients is adjusted to pH 5.5 and purified on a column of diethylaminoethyl (DEAE) cellulose which is equilibrated at pH 5.5 with 0.2 M acetate buffer. In contrast to this acid pH procedure (5.5) and in contrast to the electrofocussing used by Matsuda et al, the present invention concentrates on an agarose buffer at a pH 8 with dipotassium acid phosphate buffers. The exchange resin in the present invention is an anion exchange resin of agarose and the fractionation accomplished after discarding the first fraction and eluting the absorbed protein with a small volume of buffered phosphate of pH 8 in the presence of increasing quantities of salt (0.1 molar up to 0.2 molar) results in a purification of at least 10 to 20 fold and up to 80 fold.
The U.S. Pat. No. 3,960,827, to Bjorklund, shows a procedure for purifying a cancer associated polypeptide antigen employing various molecular sieve type gels in bead form such as polyacrylamide gels, dextran gels and agarose gels which are equilibrated with suitable buffer at pH above 7.0. In the examples of the gels given in this patent none of these are gels having covalently linked groups of diethylaminoethyl. Further the technique of Bjorkland is to select an isolectric adjustment of the pH in order to precipitate the protein. To accomplish this precipitation a pH gradient is created which is tested. This complex procedure for purification is avoided in the present invention by the use of a covalent linked agarose beads having diethylaminoethyl groups attached and in the best embodiment also having Cibacron Blue F3GA dye attached.
It is known to use peroxidase as a label for use in ELISA and examples of purification of protein followed by ELISA assay have been described in the patent literature in DeFazio, et al, U.S. Pat. No. 4,447,545 for bladder cancer detection.
DeFazio et al describe a screening of the at risk population and a detection method in which a particular protein, called CRP protein found in inflammatory disease and in metastatic cancer can be recovered from bladder cancer patients. A first step is to collect urine and to separate bladder cancer protein on a chromatographic column in which the absorbent is hydroxyapatite. Two separate fractions are eluted at different phosphate levels. The second fraction was tested against anti-CRP, an anti-serum which is (Commercially available from Miles Laboratory) in immunoelectrophoresis, at pH 8.2 in an agarose gel. In contrast, the present invention uses an anion exchange beaded agarose chromatographic column covalently linked to DEAE and preferably also to Cibacron Blue F3GA.
The second step in DeFazio is an ELISA quantitative assay.
It is also known in Muroi, et al, U.S. Pat. No. 3,725,075, to use increasing sodium chloride concentrations for elution to separate proteins. This patent is of interest for the general teaching of the principles of protein separation at optimum pH.
It is noted that the protein purification taught by DeFazio, et al, U.S. Pat. No. 4,447,545, is based upon the recovery of a much larger amount of protein which is characteristic of bladder cancer in humans than the amount of protein present in gastric juice.